HLA genes play a pivotal role in the immune response via presenting the pathogen peptides on the cell surface in a host organism. Here, we studied the association of HLA allele variants of class I  and class II  genes with the outcome of COVID-19 infection. We performed high-resolution sequencing of class HLA I and class II genes based on the sample population of 157 patients who died from COVID-19 and 76 patients who survived despite severe symptoms. The results were further compared with HLA genotype frequencies in the control population represented by 475 people from the Russian population. Although the obtained data revealed no significant differences between the samples at a locus level, they allowed one to uncover a set of notable alleles potentially contributing to the COVID-19 outcome. Our results did not only confirm the previously discovered fatal role of age or association of DRB1*01:01:01G and DRB1*01:02:01G alleles with severe symptoms and survival, but also allowed us to single out the DQB1*05:03:01G allele and B*14:02:01G~C*08:02:01G haplotype, which were associated with survival. Our findings showed that not only separate allele, but also their haplotype, could serve as potential markers of COVID-19 outcome and be used during triage for hospital admission. In 2020, humankind was threatened by the epidemic that affected almost all countries to a certain degree. It was caused by a new virus from the Coronaviridae family—SARS-CoV-2 . The firOne of the essential factors facilitating such a rapid spread of COVID-19 is a striking variability in the disease manifestations, as well as the mortality rate in individual patients ,4,5. TheThe main function of these molecules is to present an antigen on the plasma membrane surface so that it would be recognized by immune cells. Therefore, the alleles encoding the amino acid sequences in HLA directly affect the susceptibility to certain diseases, e.g., H1N1 influenza  , etc. 1,14.n = 26, R = −0.44, p-value = 0.02) [p-value = 0.003 after adjustment for age and sex) [Based on the sequencing of the alleles of HLA class I and II genes and the protein composition of SARS-CoV-2, authors analyzed the affinity level of MHC binding to all possible viral epitopes ,16. The  = 0.02) . The resand sex) . In the and sex) .1 = 1980, n2 = 332) GWA studies produced quite opposite results: one of them failed to show any association between COVID-19 and HLA genes in European population [Thus, HLA alleles may yield different results in terms of statistical significance depending on the studied population ,25,26,27pulation . Furtherpulation ,24.In our work, we sequenced six HLA loci  and class II ) to compare the frequencies of HLA alleles and HLA haplotypes in three groups: (1) healthy donors from bone marrow registry, (2) patients who survived COVID-19 despite a severe course of a disease, and (3) patients who died from COVID-19 with an adjustment for age and comorbidities. This work aims to identify and validate the alleles significantly related to various COVID-19 outcomes. Therefore, we performed a retrospective analysis in order to reveal any possible associations between the identified alleles and the disease outcomes in the Russian population. First, differences in sex, age, and allele frequencies were assessed using Fisher’s exact test and Pearson’s chi-squared test. Then, Hardy-Weinberg equilibrium and nonequilibrium linkage were evaluated for each group and each locus. After that, haplotype frequencies were assessed using the maximum likelihood method, and differences in frequencies were evaluated using Fisher’s exact test and the t-test. In the final step, logistic regression models, including sex, age, and HLA locus or allele, were constructed.Females predominated in all studied groups .Using the Pearson’s chi-squared test, we detected a statistically significant deviation between the groups .Pairwise comparisons revealed statistically significant differences between groups 1 and 3 . In group = 0.72). In the other groups, there was a marked shift toward older age  or younger age . For this reason, the non-parametric Mann-Whitney test was chosen as the statistical test.The age distribution in groups is shown in The statistically significant differences in the age were observed in all compared groups .p-value < 0.05). At the same time, we did not detect any statistically significant differences in the age of patients homozygous for class I or class II loci, class II loci (in combinations), or homozygous for at least one locus.The average age of deceased patients homozygous for at least one allele of class I loci  was lower as compared to the patients without homozygous loci , as well as HLA-C and HLA-DPB1 , was statistically insignificant. In group 3, statistically insignificant linkage was observed only between the following pairs: HLA-A and HLA-DQB1 , HLA-A and HLA-DPB1 , and HLA-C and HLA-DPB1 . Based on the obtained results, we further performed a haplotype analysis for all six loci , as well as for five loci , for the class I and II loci, and the pairs of the HLA-B and HLA-C demonstrating statistically significant deviations from the Hardy-Weinberg equilibrium.The linkage disequilibrium was more prominent in group 1. All analyzed loci in this group were related statistically significant to each other. In group 2, the linkage between the HLA-A and HLA-DPB1  or allele combination (V1) from three groups.To identify significant alleles, we studied the combinations of other groups using the V1 and V2 methods. We analyzed different group combinations to determine significant individual alleles or their pairs associated with either a good or bad outcome . The dirFinally, various combinations of groups were studied to determine significant alleles .The most frequent haplotypes estimated for five loci showed a higher convergence as compared to the analysis of six loci . The mosp-value = 0.003, t-test adjusted p-value = 0.028). Analyzing class I haplotypes  revealedp < 0.05, t-test adjusted p < 0.05; exact Fisher’s test p < 0.05, t-test adjusted p < 0.05). Comparing group 1 and 2 allowed us to single out the haplotype (DRB1*07:01:01G~DQB1*02:01:01G~DPB1*17:01:01G) with a frequency  exhibiting statistically significant differences between the groups . Comparison of the frequencies of the class II loci between groups 1 and 3 revealed the haplotype (DRB1*11:01:01G~DQB1*03:01:01G~DPB1*04:02:01G). Its frequency was statistically significant between the groups , according to the results of two tests .Comparing group 2 and 3  enabled p < 0.05, t-test adjusted p < 0.05; exact Fisher’s test p < 0.05, t-test adjusted p < 0.05). Analyzing group 1 and 2 revealed the haplotype ) with a frequency being statistically significantly different between groups . Comparing group 1 and 3 revealed the haplotype ) with statistically different frequencies between groups .Comparing the frequencies of the HLA-B and HLA-C loci  between p < 0.05) and age (p < 0.05)  .In order to avoid the effect of linkage disequilibrium and, as a consequence, the presence of strongly correlated independent variables, each locus was analyzed independently. p < 0.05), intercept (p < 0.05), and age (p < 0.05). We selected the best model by gradually excluding the predictors based on the AIC parameter and observed a loss of statistical significance by the allele.In our logistic model, including only the locus HLA-A alleles, revealed statistical significance of the A*33:01:01G allele (p < 0.05), HLA-B*35:03:01G (p < 0.05), HLA-B*38:01:01G (p < 0.05), HLA-B*40:01:01G (p < 0.05), HLA-B*57:01:01G (p < 0.05) alleles, and age (p < 0.05). After selecting the best model by gradually excluding the predictors based on the AIC parameter, along with the predictors mentioned above, we identified significant differences in the frequencies of HLA-B*07:02:01G (p < 0.05), HLA-B*13:02:01G (p < 0.05), HLA-B*18:01:01G (p < 0.05), HLA-B*41:01:01G (p < 0.05), HLA-B*44:03:01G (p < 0.05), and HLA-B*56:01:01G (p < 0.05). A separate model, including a single allele, sex, and age, was statistically significant only in case of the HLA-B*14:02:01G allele (p < 0.05).Using the logistic model based only on the HLA-B locus alleles, observed, statistical significant predictors included HLA-B*14:02:01G (p < 0.05), age (p < 0.05), and the HLA-C*08:02:01G allele (p < 0.05). After selecting the model by gradually excluding the predictors based on the AIC parameter, we identified additional significant predictors: HLA-C*12:02:01G, HLA-C*15:02:01G, and the interaction of sex and age   . The modThe logistic model including only the HLA-DQB1 locus alleles did not reveal statistically significant determinants apart from the age. After selecting models by gradual exclusion of predictors based on the AIC parameter, statistical significance was detected in case of the HLA-DQB1*05:01:01G allele . The modThe logistic model including the DPB1 locus alleles did not reveal any statistically significant differences, except the age, for all alleles in this locus as well as for individual alleles.We also designed the model based on all identified statistically significant alleles HLA-C*08:02:01G (HLA-B*14:02:01G was removed due to 100% correlation with HLA-C*08:02:01G), HLA-DRB1*01:01:01G, and HLA-DQB1*05:01:01G, as well as sex, age, and their interaction. After selecting the models by gradually excluding the predictors based on the AIC parameter, statistically significant predictors included only DRB1*01:01:01G and HLA-C*08:02:01G .The group of recovered patients (group 2) with severe symptoms, and the patients who died from COVID-19 (group 3) were divided into subgroups  according to the age (age < 65 or ≥ 65 at the moment of death/illness). Clinical features of the groups are presented in The exploited biomaterial consisted of venous whole blood collected into EDTA-coated tubes. Diagnostic criteria for inclusion to the study were fever and/or respiratory symptoms and the positive test for COVID-19 was confirmed by RT-qPCR test –named «SARS-CoV-2/SARS-CoV» —from nasopharyngeal swabs in Moscow clinical diagnostic laboratories that collected the biomaterial. Patients with pathologies that led to greater morbidity or who had additional immunosuppression  were not included in the study.gDNA was isolated from 100 uL of venous whole blood with the Proba-McheMaks  reagent kit using the automated dosing station DTstream . This method involved a routine step including lysis, DNA precipitation on magnet beads, three washing steps, and an elution step. Quality control of the isolated DNA was performed by agarose gel electrophoresis; the concentration was measured using Qubit 3 fluorometer with Qubit dsDNA BR Assay kit  .The preparation of amplicon libraries for HLA high-resolution genotyping was performed using HLA Expert kit  following the manufacturer’s protocol ). It included several steps. The first stage involved a qPCR for human gene that does not have pseudogenes and is presented in a single copy. This was required for the estimation of a concentration and the presence of inhibitors in a genomic DNA sample. The results were used for normalization of DNA amount during the following step. The second stage involved a multiplex PCR for most variable exons . Primers were designed using conservative regions of gene introns flanking the exons. Several primers with one nucleotide shift were used to prevent an imbalance in nucleotide content during sequencing. The third stage involved ligation of the adapters containing Illumina i5 and i7 indexes. The fourth stage was an additional routine PCR (6 cycles) with the p5 and p7 primers. The purification with magnetic beads (SPRI type) was performed after each stage. Quality control of the libraries was performed using agarose gel electrophoresis; the concentration was measured using the Qubit 3 fluorometer with the Qubit dsDNA HS Assay kit .Sequencing was performed using the Illumina MiSeq platform  with the MiSeq Reagent Kit v3 (600-cycle), according to the manufacturer’s protocol.Fastq files were analyzed with HLA-Expert software  following the manufacturer’s instructions. Obtained exon sequences were aligned to the human major histocompatibility complex (MHC) sequences IMGT/HLA v3.41.0 . Quality threshold for reads ;Lowest absolute and relative coverage for each position;The highest number of differences  from the group average for each read;Maximum relative position error—the number of differences  from the consensus sequence in each position should not exceed the specified threshold;The highest average error per read for a group;The lowest number of reads in groups for each exon ;The allelic imbalance should not exceed a given threshold; the ratio of the read number for the exons from each allele and the sum of these ratios;The presence of phantom (cross-mapping) and chimeric sequences;The percentage of combined, clustered, and used for typing reads computed for each sample.Basic quality control metrics for QC included:https://github.com/genomecenter/HLA_article; accessed on 22 June 2021). We created a script that generated an input file containing patients’ data for Arlequin.Allele frequencies in the analyzed cohorts were estimated by dividing the number of occurrences of a given allele in an individual by the doubled total number of individuals . Statistical analysis included the Pearson’s goodness-of-fit test , the Fisher`s exact test for determining the significances in differences between allele frequencies, the Wilcoxon rank sum test with continuity correction for estimating the differences in age between all groups. Arlequin (version 3.5.2.2) was used to conduct population assignment test, estimate the Hardy-Weinberg equilibrium, pairwise linkage disequilibrium, and measure the distances upon pairwise comparisons between all three groups . We crean = 1000). The differences of mean frequencies of haplotypes between samples were compared with the t-test with mean haplotype frequencies and standard deviation (the number of identified haplotypes in samples were used as freedom degrees). After that, we checked the results by the Fisher’s exact test.In order to determine the significant alleles for each gene by compiling a contingency matrix, we used the Holm-Bonferroni method  with theWe employed the Pearson’s goodness-of-fit test to study each gene separately and analyzed the 2-field level of alleles for each gene. The null hypothesis stated that HLA did not affect the divergence of allele distribution and allele frequencies in the groups. For evaluating the role of single alleles and allele combinations, they were selected from the groups by two methods. The contingency matrix for each gene was compiled using one of two methods . The firWe used logistic regression to discern the impacts of age and sex from the influence of alleles. We employed two models. The first model included only sex and age as independent variable and an outcome as a dependent variable. The second one used sex, age, and the presence of a certain allele as an independent variable and an outcome as a dependent variable. Analysis was conducted with glm and step from the R stats package (ver 4.2.0).Fisher’s exact test (R language):Pearson’s chi-square test (Python 3.9 language):Unequal variances t-test (R language):D—probability function, p—given haplotype frequencies) (Arlequin):Haplotype frequency estimation (Logistic regression formula (R language):Since the HLA genotype determines an individual repertoire of immune response to foreign pathogens, it could contribute to COVID 19 susceptibility and severity. Particularly, it is of importance to analyze certain alleles and haplotype frequencies in detail across different populations.Several studies see  suggestep < 0.05), group 1 and group 3A (p < 0.05), which is in line with the previous results [In our work, we scrutinized three groups comprising the representatives of the Russian population. We used various approaches for estimating the link between individual HLA alleles and generated haplotypes with the COVID-19 outcome. We found a significant bias in the sex and age composition of the population. Group 1 mostly consisted of females (71.2%), whereas group 3 included mostly males (56.1%). This may be explained by the statistically significantly association of the sex with the survival rate after severe disease as it was shown previously [Our findings reveal that due to linkage disequilibrium, the statistically significant alleles were combined into individual haplotypes that could predominate in the populations of deceased patients (B*27:02:01G~C*02:02:02G) or survivors (B*14:02:01G~C*08:02:01G). The obtained results are only partially consistent with the previous findings , which can be related to the population characteristics, as well as with the insufficient sample size that greatly decreased the statistical power of the analysis. Another decline in the analysis efficiency might arise from the possibility that, in the population, the alleles with high affinity toward viral peptide  can be co-inherited with the alleles showing the lower affinity  (Ref. ). ExceptThe main limitation of this work is the lack of detailed clinical data for patients, which does not allow for a study of the relationship between the analysis of HLA genotype or haplotypes and additional factors affecting survival, such as comorbidities, BMI, etc. Another important limitation is that the relatively small sample size, which could significantly reduce the power of the assessment methods used, especially for rare alleles and haplotypes.In the present study, we analyzed HLA genotypes of three Russian population samples: healthy individuals, patients who survived severe COVID-19, and patients who died from it. Using the Fisher’s exact test and Pearson’s goodness-of-fit test, we performed haplotype frequencies analysis an logistic regression to show that the alleles of loci A, B, C, DRB1, DQB1 and DPB1 influenced the COVID-19 outcome. The immediate results of the research showed the absence of any significant difference between the groups at the locus level, however, several alleles proved to be perspective. These embrace the already known DRB1*01:01 and DQB1*05:03, detected in the current research, since they presumably influence the outcome of COVID-19. We also found a decrease in the frequency of one of the common haplotypes (B*14:02:01G~HLA-C*08:02:01G) in the group of deceased patients. On the contrary, the frequency of this haplotype in the group of survivors three times exceeded its occurrence in the control group. Still, the results allow us to conclude that the associations of HLA alleles with COVID-19 progression and outcome depend largely on individual characteristics of the population under investigation. In further work, we plan to collect samples of larger size and more detailed information on comorbidities, which will allow to obtain higher power for statistical criteria, as well as to make a more accurate assessment of the role of HLA genes and their haplotypes in the course of the disease.


An update of a systematic review and meta-analysis of the risk of arrhythmias and their subtypes in type 2 diabetic patients receiving glucagon-like peptide 1 receptor agonist (GLP-1RA) medication according to data from the Cardiovascular Outcome Trial(CVOT).Randomized controlled trials (RCT) on GLP-1RA therapy and cardiovascular outcomes in type 2 diabetes mellitus patients published in full-text journal databases such as MEDLINE (via PubMed), Embase, Clinical Trials.gov, and the Cochrane Library from establishment to March 1, 2022 were searched. We assessed the quality of individual studies by the Cochrane risk-of-bias algorithm. RevMan 5.4.1 software was use for calculating meta-analysis.p =0.36], and its subtypes such as atrial fibrillation , atrial flutter , atrial tachycardia , sinoatrial node dysfunction , ventricular preterm systole , second degree AV block , complete AV block , ventricular fibrillation , ventricular tachycardia  from treatment with GLP-1RA versus placebo. However, the risk of hypoglycemia was reduced by about 30%  and the risk of pneumonia by about 25% , both statistically significant differences.A total of 60,081 randomized participants were included in the data of these 8 GLP-1RA cardiovascular outcomes trials. Pooled analysis reported no significant effect on total arrhythmia [RR=0.96, 95% CI , In type 2 diabetic patients, treatment with GLP-1RA has no significant effect on the risk of major arrhythmias but significantly reduces the risk of hypoglycemia and pneumonia. Diabetes is one of the most common chronic diseases worldwide, and cardiovascular events are the most major complications in type 2 diabetics, including atrial and ventricular arrhythmias , 2. The GLP-1RA is a new type of glucose-lowering drug that lowers HbA1c, modestly improves blood lipids and body weight, and decreases the risk of hypoglycemia by stimulating insulin secretion and lowering glucagon secretion, delaying gastric emptying, and reducing appetite . It has The key role of the cardiac ANS in the development of arrhythmias has been confirmed by research that began as early as 60 years ago . From thp < 0.00001, I2 = 0%, random-effect modelling)  . On thisTherefore, the intention of our analyses was to explore the impact of GLP-1RA treatment on the occurrence of several different types of arrhythmias in type 2 diabetic patients based on data extracted from relevant cardiovascular outcome trials, with the addition of some secondary indicators of interest, such as hypoglycemia and pneumonia cases.The English and Chinese literature on GLP-1RA associated with cardiovascular outcomes published since the establishment of the database until March 1, 2022 was searched manually by computer in MEDLINE (via PubMed), Embase, Clinical Trials.gov, and Cochrane Library databases. Search terms: “Glucagon-like peptide-1 receptor agonist”, “Cardiovascular”, “ cardiac failure”, “lixisenatide”, “exenatide”, “liraglutide “, “Semaglutide”, “Albiglutide”, “Dulaglutide”, “Efpeglenatide”, “placebo”, “Clinical Trials”.Inclusion criteria: (1) Randomized, double-blind, parallel-group, multicenter study of clinical trials; (2) Patients with type 2 diabetes who are at high cardiovascular risk  are the primary study subjects; (3) Intervention with GLP-1 receptor agonist and control with placebo; (4) Data on arrhythmias, hypoglycemia, and pneumonia must be available in all trials for adverse events; (4) A more complete table of baseline patient characteristics is available; (5) Published English literature up to March 1, 2022.Exclusion criteria: (1) Reviews, reports, and conference proceedings on GLP-1RA and cardiac arrhythmias; (2) Studies with inaccessible full text or incomplete data; (3) Repeatedly published or repeatedly included studies or studies with similar information; (4) Clinical trials that included type 1 diabetic patients.Total arrhythmias and major arrhythmia types .hypoglycemia (defined as diabetes mellitus in patients receiving medication as long as their blood glucose is below 3.9 mmol/L) and pneumonia .Randomized controlled trials comparing GLP-1RA with placebo in type 2 diabetic patients at high cardiovascular risk were included. Outcomes of interest included atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular tachycardia, atrial tachycardia, sinus node dysfunction, ventricular asystole, second degree AV block and complete AV block, hypoglycemia, and pneumonia. First, titles and abstracts were screened to assess their potential eligibility for inclusion, and then full-text checks were applied to determine final eligibility. The following information was collected using a predefined data extraction form: study information , patient characteristics , therapy information  and outcome data (number of events per outcome). All outcomes of interest were dichotomous, first preferentially extracting data from ClinicalTrials.gov and secondarily selecting data from the original trial publication or secondary analysis of the same trial. The Cochrane Risk of Bias Tool was used to assess the quality of included studies . Bias wap <0.05 being a statistically significant difference. Heterogeneity analysis among groups between studies was executed using the χ2 test, and the results are presented as 2I. Fixed-effects models were used for analysis if there was homogeneity among studies (p >0.05 or 2 ≤I 50%), and random-effects models were used for analysis if there was heterogeneity among studies (p ≤ 0.05 and 2I>50%). Sources of heterogeneity can be searched for by sensitivity analysis and subgroup analysis when large heterogeneity exists. Due to the number of trials being less than 10, publication bias was not evaluated.Data were analyzed with RevMan 5.4.1, and effect analysis statistics were expressed as RR and 95% CI, 2008 literatures were initially screened in the database according to keywords, and 176 were obtained after de-duplication and exclusion of review literature, irrelevant literature, incomplete data, unreported cardiovascular events, unspecified results, and incorrect study type, and then 8 clinical trials with a total of 68,001 patients were finally included after the screening process was repeated by two investigators again Figure 12 =I44%, p =0.08), atrial flutter , ventricular fibrillation , ventricular tachycardia , atrial tachycardia , sinus node , ventricular precontraction , second degree AV block , and complete AV block , so a fixed-effect model was used to combine effect sizes.Separate data-analyses were estimated according to the type of adverse event arrhythmia disclosed, homogeneity of included studies was good for all events, with the following results:atrial fibrillation , so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on total arrhythmias outcomes from treatment with GLP-1RA versus placebo .The eight studies collected for mate-analysis of the effect of GLP-1RA on total arrhythmias are listed in 2=I44%, p=0.08), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on atrial fibrillation outcomes from treatment with GLP-1RA versus placebo .The eight studies collected for mate-analysis of the effect of GLP-1RA on atrial fibrillation are listed in 2=I0%, p=0.49), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on atrial flutter outcomes from treatment with GLP-1RA versus placebo .The seven studies collected for mate-analysis of the effect of GLP-1RA on atrial flutter are listed in 2=I0%, p=0.86), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on ventricular fibrillation outcomes from treatment with GLP-1RA versus placebo .The five studies collected for mate-analysis of the effect of GLP-1RA on ventricular fibrillation are listed in 2=I0%, p=0.42), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on ventricular tachycardia outcomes from treatment with GLP-1RA versus placebo .The seven studies collected for mate-analysis of the effect of GLP-1RA on ventricular tachycardia are listed in 2=I0%, p=0.45), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on atrial tachycardia outcomes from treatment with GLP-1RA versus placebo .The four studies collected for mate-analysis of the effect of GLP-1RA on atrial tachycardia are listed in 2=I0%, p =0.54), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on Sinus Node Dysfunction outcomes from treatment with GLP-1RA versus placebo .The seven studies collected for mate-analysis of the effect of GLP-1RA on Sinus Node Dysfunction are listed in 2=I0%, p =0.64), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on ventricular asystole outcomes from treatment with GLP-1RA versus placebo .The six studies collected for mate-analysis of the effect of GLP-1RA on ventricular asystole are listed in 2=I0%, p =0.84), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on second-degree AV block outcomes from treatment with GLP-1RA versus placebo .The six studies collected for mate-analysis of the effect of GLP-1RA on second-degree AV block are listed in 2=I40%, p =0.13), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported no significant effect on complete AV block outcomes from treatment with GLP-1RA versus placebo .The seven studies collected for mate-analysis of the effect of GLP-1RA on complete AV block are listed in 2=I33%, p =0.18), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported statistically significant effect on hypoglycemia outcomes from treatment with GLP-1RA versus placebo , showed that the risk of hypoglycemia was about 30% lower in the GLP-1RA treatment group than the placebo group.The seven studies collected for mate-analysis of the effect of GLP-1RA on hypoglycemia are listed in 2=I50%, p =0.06), so the fixed-effects model was used to combine the effect sizes. Pooled analysis reported statistically significant effect on hypoglycemia outcomes from treatment with GLP-1RA versus placebo , showed that the risk of pneumonia was approximately 25% lower in the GLP-1RA treatment group than in the placebo group.The seven studies collected for mate-analysis of the effect of GLP-1RA on pneumonia are listed in p =0.36] (p =0.43] (p =0.30] (p =0.44)] , p =0.36] Figure 2p =0.36] in patients with type 2 diabetes. Pooled data suggest that GLP-1RA treatment has no effect on the risk of various types of arrhythmias. The risk of hypoglycemia was statistically significantly reduced by approximately 30%  and pneumonia by approximately 25% . Based on the differences in primary and secondary outcome results seen in our analysis, we speculate that the neutral effect of GLP-1RA on arrhythmias may be due to the pharmacological effects of GLP-1RA itself, but may also be related to a reduction in the risk of hypoglycemia and thus some decrease in the sympathetic excitatory effects of stressful events。The results of the Cochrane risk of bias assessment among the included studies were of high quality, with low publication bias and good homogeneity, so no further sensitivity analysis was required. Compared with other CVOTs involving GLP-1RA, participants in the AMPLITUDE-O trial had the longest duration of diabetes (15 years), the lowest mean eGFR (72 ml/min/1.73 m 2), a higher proportion of nephropathy (eGFR <60 mL/min) at baseline (32%), the highest glycated hemoglobin (8.9%), and the highest percentage of sodium -glucose transporter 2 inhibitor (SGLT-2i) use (15%), suggesting that the AMPLITUDE-O trial was in a population with poorer underlying conditions  may increase oxidative stress and promote innate or adaptive immune dysfunction, leading to an increased chance of infection . Therefop=0.006] and no significant heterogeneity was observed between subgroups of individuals with or without cardiovascular disease (p=0.127). It also reduced the risk of cardiovascular death by 13% (p=0.016), non-fatal stroke by 16% (p=0.007), hospitalization for heart failure by 10% (p=0.023), and all-cause mortality by 12% (p=0.012) (6). However, there appears to be a neutral effect on the risk of arrhythmia. Our findings suggest that GLP-1RA may has no effect on the risk of most types of arrhythmias compared with placebo. It demonstrates that despite the sympathetic excitatory effect of GLP-1RA, GLP-1RA exhibits a degree of safety in terms of the risk of arrhythmia in patients with type 2 diabetes, with the implication that the use of GLP-1RA analogues may be considered to improve or maintain glucose-lowering therapy in patients with a long history of disease when clinically encountering patients with arrhythmias that meet the indications but for whom the use of SGLT2i is contraindicated. From another perspective, our findings serve as reverse evidence that further positively supports the consistent results presented by numerous international studies so far: GLP-1RA treatment can indeed bring cardiovascular benefits and safety to diabetic patients.Despite a large body of scientific research, GLP-1RA demonstrated a positive effect with a 14% reduction in major cardiovascular events (MACE) [HR=0.86, 95%CI(0.79-0.94), Despite our comprehensive systematic evaluation of the RCTs of GLP-1RA, some limitations remain. First, none of the included trials were specifically designed to assess arrhythmic outcomes with GLP-1RA, and all trials were designed to assess the safety of GLP-1RA. Further validation could be done in future by designing large clinical studies with arrhythmia as an endpoint. Second, different chemical structures, pharmacological profiles, and modes of action of these GLP-1RA are available, and although the differences are subtle, head-to-head cardiovascular outcomes trials for this class of drugs are not available, which limits the ability to detect differences in outcomes between GLP-1 agonists of different structures or potencies and to rule out whether they affect treatment outcomes. Third, differences in trial populations including baseline data could also be a potential explanation for differences in study outcomes. Nevertheless, we only collected overall data in our analysis, which limited our capability to further explore any subgroups of interest, to group trials according to combinations of disease processes, such as SGLT2i with GLP-1RA, amount of atrial fibrillation events in patients with or without pre-existing heart failure, and understanding of missing data remains to be explored. Therefore, the findings of the subgroup analysis have to be interpreted cautiously and further investigations are needed to assess whether GLP-1RA may reduce the incidence of arrhythmias in patients with multiple comorbidities. Fourth, the follow-up time of the study was also one of the limitations. Although these studies are considered long-term randomized controlled trial studies, they still only cover roughly 2-4 years, and patients may have been using GLP-1RA in clinical practice for a very short period of time. It is still not possible to exclude additional benefits or damages that may arise from shorter or longer exposure times. Fifth, the prevalence of these combined treatment effect sizes is determined by the study population, which is characterized by low insulin secretory capacity, insulin resistance and mild obesity relative to the American group, and regional subgroup analysis may provide more informative information if necessary. Sixth, no animal or cell-based experiments were focused on in this study. The above limitations may weaken the persuasiveness of our study, but the majority of eligible studies selected by 2 different investigators based on strict inclusion criteria were of high quality through a comprehensive literature search of 4 databases. Therefore, we believe it is reasonable to draw conclusions from this meta-analysis.Overall, our study showed a neutral effect of GLP-1RA on the risk of major arrhythmias. However, it exactly in turn support the favorable effect of GLP-1RA on cardiovascular outcomes in patients with type 2 diabetes. The secondary outcomes of hypoglycemia and pneumonia showed statistically significant risk reduction, which provides further rationale for the safety of GP-1RA treatment. However, additional randomized clinical trials are needed in the future to confirm this conclusion.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JW designed the research, JW and RWcontributed to the literature database search, data collection, data extraction, data analysis, and writing of the manuscript. JW, RW, HY and YW participated in the discussion. XZ and LW reviewed and revised this article. All authors contributed to the article and approved the submitted version.This study was supported by Talent introduction funding project of the First Affiliated Hospital of Jinan University (no.808026) and Basic Scientific Research Project of central Universities of Jinan University (no.21622301).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


All-polymer solar cells (All-PSCs), whose electron donor and acceptors are both polymeric materials, have attracted great research attention in the past few years. However, most all-PSC devices with top-of-the-line efficiencies are processed from chloroform. In this work, we apply the sequential processing (SqP) method to fabricate All-PSCs from an aromatic hydrocarbon solvent, toluene, and obtain efficiencies up to 17.0%. By conducting a series of characterizations on our films and devices, we demonstrate that the preparation of SqP devices using toluene can effectively reduce carrier recombination, enhance carrier mobility and promote the fill factor of the device. With the increasing focus on new energy technology, organic solar cells (OSCs) have become one of the hottest research directions in the field of thin film photovoltaics . In addiWhen the electron donor is a polymer and the non-fullerene acceptor is also a polymer, the solar cell is typically referred to as an all-polymer solar cell (All-PSC) ,17. Due γ, we, for the first time, obtained All-PSCs devices with an efficiency of 17.0% using toluene as the main solvent. Through film-depth-dependent light absorption spectra (FLAS) experiments, we found that vertical phase segregation can be significantly improved by the SqP method, which leads to a large increase in the FF and, thus, the efficiency of the device. In order to further investigate the device physics, we carried out transient photovoltage (TPV) and transient photocurrent (TPC), Fourier-transform photocurrent spectroscopy external quantum efficiency (FTPS-EQE), light intensity dependent J-V measurement, etc., which revealed that the SqP method can effectively reduce charge recombination, increase and balance charge transport and enhance the charge carrier lifetime.In this work, we used toluene (Tol), an aromatic hydrocarbon solvent which is less toxic and has a boiling point as high as the solvent, to make all-PSC devices. In order to optimize both the bulk morphology and vertical phase segregation, we use the sequential processing (SqP) technique to prepare the active layer instead of the BC method. SqP, also called layer-by-layer (LBL), quasi-bilayer, pseudo-bilayer, etc. in the literature, has been shown to be an effective method to prepare the active layer of organic solar cells in both small and large areas with enhanced performance, batch-to-batch consistency and even stability ,31. A deγ and PNDIT-F3N were purchased from eFlexPV Limited . PEDOT:PSS (Clevios P VP 4083) was purchased from Heraeus Inc. . All the other reagents and chemicals were purchased from Sigma Aldrich or Aladdin and used as received.PM6 was purchased from Solarmer Material Inc. . PY-V-Device fabrication. Solar cells were fabricated in a conventional device configuration of ITO/PEDOT:PSS/active layer/PNDIT-F3N/Ag. The ITO substrates were cleaned by detergent and then sonicated with deionized water, acetone and isopropanol, and dried overnight in an oven. The glass substrates were treated with UV-Ozone for 30 min before PEDOT:PSS was spin-casted on top at 5000 rpm for 30 s, and then annealed at 150 °C on a hotplate for 10 min in air.(1)γ (BC), the PM6: PY-V-γ blend (1:1.2 weight ratio) was dissolved in toluene , with 1-chloronaphthalene (1% vol) as an additive and stirred overnight in a nitrogen-filled glove box. The 95 °C toluene blend solution was spin-casted at 2500 rpm for 30 s onto the PEDOT:PSS films followed by a thermal annealing of 95 °C for 5 min.For the blend-casting (BC) devices, namely, PM6:PY-V-(2)γ (SqP), PM6 was dissolved in toluene (the concentration of donor was 8 mg mL−1); PY-V-γ was also dissolved in toluene (the concentration of donor was 12 mg mL−1) but with 1-chloronaphthalene (2% vol) as an additive. Both solutions were stirred overnight in a nitrogen-filled glove box. The donor solution was spin-casted at 4000 rpm for 30 s onto the PEDOT:PSS films, then the acceptor solution was spin-casted at 4000 rpm for 30 s onto the donor films followed by a thermal annealing of 95 °C for 5 min.For the sequentially processed (SqP) device, namely, PM6/PY-V-BC and SqP devices: −1 was spin-coated onto the active layer at 2000 rpm for 30 s. Around 100 nm of Ag was evaporated under 4 × 10−4 Pa through a shadow mask. Then, the encapsulation was carried out.For all types of devices, a methanol with 0.5% vol acetic acid blend solution of PNDIT-F3N at a concentration of 0.5 mg mLDevice characterization. The details of J-V testing, and EQE measurements are shown in the SCLC Measurements: The electron- and hole-mobilities were evaluated using the space-charge limited current (SCLC) method. The device architecture of the electron-only devices was ITO/PNDIT-F3N/active layer/PNDIT-F3N/Ag and that of the hole-only devices was ITO/PEDOT:PSS/active layer/ Au. The charge carrier mobilities were determined by fitting the dark current into the model of a single carrier SCLC according to the equation: J = 9ε0εrμV2/8d3, where J is the current density, d is the film thickness of the active layer, μ is the charge carrier mobility, εr is the relative dielectric constant of the transport medium, and ε0 is the permittivity of free space. The V used in the equation is defined by: V = Vapp –Vbi, where Vapp is the applied voltage, Vbi is the built-in voltage. The carrier mobilities were calculated from the slope of the J~V2 curves.FTPS-EQE: FTPS-EQE measurements were conducted on the same devices used in J-V measurements using an integrated system (PECT-600) purchased from Enli Technology Co., LTD . The range of measurements was 600–1500 nm. The signal due to noise was cut out for all devices below 0.83–2.07 eV. Film-depth-dependent light absorption spectroscopy (FLAS) and composition distribution: Film-depth-dependent light absorption spectra were acquired by an in situ spectrometer   equipped with a soft plasma-ion source. The power-supply for generating the soft ionic source was 100 W with an input oxygen pressure ~10 Pa. The film surface was incrementally etched by the soft ion source, without damage to the materials underneath the surface, which was in situ monitored by a spectrometer. From the evolution of the spectra and the Beer–Lambert’s Law, film-depth-dependent absorption spectra were extracted. The composition distribution along the film-depth direction was obtained from the film-depth-dependent spectra by fitting the sub-layer absorption using the absorption of the pure components. The exciton generation contour is numerically simulated upon inputting sub-layer absorption spectra into a modified optical transfer-matrix approach. The detailed experimental and numerical method are available elsewhere ,34.γ are shown in γ as the active layer materials were fabricated, named PM6:PY-V-γ (BC) and PM6/PY-V-γ (SqP). The final overall structure of the device is ITO/PEDOT:PSS/active layer/PNDIT-F3N/Ag, where PEDOT:PSS and PNDIT-F3N are the hole and electron transport layer, respectively. The chemical structures of the polymers, the energy levels and device fabrication schematic are shown in γ. Another peak at 400 nm–700 nm is from the absorption of PM6, while that in the range of 700–900 nm is the absorption of PY-V-γ. The blend covers the majority of the solar spectrum thanks to the complementary absorption between the donor and the acceptor. The current density and voltage (J-V) characteristics of these two devices are shown in VOC of 0.913 V, a JSC of 24.28 mA cm−2, and an FF of 0.733. Then, the device fabricated with SqP method shows an elevated FF (0.764), which resulted in the highest PCE of 17.0%. These results demonstrate that the voltage and current for the devices based on BC and SqP do not differ significantly, while the main difference comes from the FF. The significantly enhanced FF from 0.733 to 0.764 leads to the efficiency improvement from 16.3% to 17.0%. More spin-coating conditions were tested, and the corresponding device J-V characteristics can be found in the JSC mainly arises from the EQEs in the range of ~680–800 nm, which is consistent with our previous observation [JSCs of the device is consistent with the integrated value from the EQE curves, with an error within 5%. ervation . CombineJSC with respect to light intensity is plotted in S) obtained from linear fittings is listed in S values for the BC and SqP devices are 0.98 and 0.99, respectively, indicating rather similar bimolecular recombination. Next, by fitting the VOC versus ln(I) result, ideality factors (nid) can be calculated, which has been shown to relate to trap-assisted recombination based on the diode theory. A higher nid indicates more trap-assisted recombination [VOCs on light intensity for the SqP and BC devices, and nid,l) under different light intensities can by calculated by I is the light intensity, q the elementary charge, k the Boltzmann constant, and T the Kelvin temperature. Compared to the BC device which shows a nid,l of 1.25, the nid,l of SqP devices is 1.11, implying less trap-assisted recombination and the carrier recombination type is mainly bimolecular (S close to 1). To further verify the ideality factor between SqP and BC, we fit the exponential region of the dark J-V curves, where the dark ideality factor  can be calculated by q, k, T, V, and J are the fundamental charge, Boltzmann constant, bias voltage and dark current density, respectively. The dark J-V curves are plotted in nid,d calculated using the slope of the linear region  model, hole-mobility (μh) and electron-mobility (μe) can be calculated. As shown in −4 cm2 V−1 s−1) and BC devices (3.61 × 10−4 cm2 V−1 s−1) are similar, but electron mobilities increase from 2.28 × 10−4 cm2 V−1 s−1 (BC) to 2.28 × 10−4 cm2 V−1 s−1 (SqP). This increase makes the SqP device more balanced in charge transport, consistent with its high FF. Next, to study the carrier lifetime and carrier extraction time, we carried out transient photovoltage (TPV) and transient photocurrent (TPC) experiments. The normalized TPV and TPC curves are shown in To assess the charge transport properties of different devices, we fabricated hole-only and electron only devices and plotted the γ, tends to segregate toward the bottom (anode) in the BC device. Such more-acceptor-less-donor distribution in the bottom part of the device is unfavorable, which could induce more surface recombination and block holes from collecting by the anode. In contrast, SqP film shows a much-improved vertical phase segregation, where the large fraction of PY-V-γ is much reduced near the bottom and that the fraction of PY-V-γ at the top surface is higher than that in the BC method. This improved the vertical phase separation, can reduce surface recombination and thus be another factor for the high FF of the SqP device. After testing FLAS for different active layer thicknesses, as shown in γ and less PM6 at the top surface of the SqP film than the BC film, which is consistent with the FLAS result, confirming the better vertical phase separation for the SqP film. Furthermore, a root mean square (RMS) roughness of 1.34 nm of the BC device is observed, close to the RMS roughness of pure PM6. In contrast, the RMS roughness of 1.59 nm for SqP device and the RMS = 1.82 nm for pure PY-V-γ indicate that there may be more acceptor PY-V-γ on the upper surface of the SqP device, further confirming the visual results.After analyzing the device physics, we turn to study the morphology. One of the initial motivations of using SqP was that it could induce better vertical phase separation. To probe it, we performed film-depth-dependent light absorption spectroscopy (FLAS). This technique utilizes low-pressure plasma to incrementally etch the film from the top surface (electron transport layer/active layer interface) to the bottom of the active layer (hole transport layer interface). By measuring the underlayer absorption in situ, FLAS can provide the absorption of the sub-layers. Fitting the sub-layer absorption with the pure component absorption, the composition distribution for each sublayer can be obtained. γ, the SqP method allowed us to process the active layer from toluene, a greener and more industry-compatible solvent than chloroform, and obtain an excellent PCE of 17.0%. In comparison, the toluene processing provides an efficiency of 16.3% for the device processed from the conventional BC method. The characterization showed that SqP method effectively reduced the carrier recombination, balanced charge transport, improved the vertical phase segregation and thus improved the FF (0.764) and PCE of the device. At the same time, the SqP method is able to separately modulate the donor and acceptor layers, making it easier to optimize the morphology of the active layer, resulting in a significant increase in electron mobility. These advantages significantly promote the significance of the SqP method in all-PSCs and the use of the SqP method in toluene solvent has far-reaching implications for future large-area fabrication of organic solar cells.In conclusion, in the all-polymer material system of donor PM6 and acceptor PY-V-


In this study, we have developed a novel hybrid tri-modality system that includes two molecular imaging methods: positron emission computed tomography (PET) and fluorescence molecular imaging (FMI) and the anatomic imaging modality X-ray computed tomography (CT). The following paper describes the system development. Also, its imaging performance was tested in vitro (phantom) and in vivo, in Balb/c nude mice bearing a head and neck tumor xenograft treated with novel gene therapy [a new approach to the delivery of recombinant bacterial gene (IL-24-expressing strain)]. Using the tri-modality imaging system, we simultaneously monitored the therapeutic effect, including the apoptotic and necrotic induction within the tumor in vivo. The apoptotic induction was examined in real-time using an 18F-ML-10 tracer; the cell death was detected using ICG. A CT was used to evaluate the anatomical situation. An increased tumor inhibition (including tumor growth and tumor cell apoptosis) was observed in the treatment group compared to the control groups, which further confirmed the therapeutic effect of a new IL-24-expressing strain gene therapy on the tumor in vivo. By being able to offer concurrent morphological and functional information, our system is able to characterize malignant tissues more accurately. Therefore, this new tri-modality system (PET/CT/FMI) is an effective imaging tool for simultaneously investigating and monitoring tumor progression and therapy outcomes in vivo.Multimodality imaging is an advanced imaging tool for monitoring tumor behavior and therapy  Cancer is still one of the major causes of death worldwide. Global cancer rates are expected to reach 21.6 million new cases per year by 2030 , one of in vivo studies reported using 99mTc-, 125I-, 18F-labelled annexin V for investigating cell apoptosis in vivo (18F-ML-10) has been developed, which was specifically designed to detect apoptotic tissues in vivo , has proved to be a very useful technique for whole-body imaging in small animals such as mice  for head and neck squamous cell carcinoma in vivo and discovered that B. breve-IL24 offers a novel, safe, and clinically acceptable therapeutic approach for tumor therapy  was developed. Using the system, we simultaneously monitored the therapeutic effect of a novel gene therapy , including the apoptotic and necrotic induction within the tumor proteins –21 localThe tri-modality imaging system has a modular design. The PET, CT, and FMI modules were initially developed separately and then integrated into the same gantry. The system includes five major components: gantry, CT, PET detector ring, FMI module, and the animal bed Figure 1The CT scanning geometry uses a rotated source-detector pair when imaging living animals. It consists of a flat-panel CMOS detector  and an X-ray tube  with a 33 μm focal spot. The X-ray tube and the CMOS detector are mounted on a high precision translation stage which is mounted on the rotatable gantry. The PET module is made up of 22 detector modules arranged in a ring-like geometry with an interior diameter of 100 mm and an axial FOV of 32 mm. The FMI module included two common fluorescence imaging modes: fluorescence molecular tomography  and fluorescence reflectance imaging . Both imaging modes rely on a tungsten-halogen lamp  as an illumination source, while an sCMOS  is used as the detection device. The whole setup is mounted on a surface plate which is also connected to the gantry module.FMI and CT imaging modules share one rotation gantry. In addition, all three modalities (PET/CT/FMI) images were scanned on the same animal bed. The animal bed was mounted on a high precision motor-controlled translation stage which carries the imaging objects along with the different imaging modalities. Multi-modal image fusion was precisely done by co-registered positional data overlay. An anesthetics vaporizer and thermostatic equipment were reserved during the experiment for animal safety reasons.-1 and a reduced scattering coefficient of µs = 10.0 cm-1, which perfectly mimics tissue properties. The phantom images were used to calibrate the co-registration of the images obtained from the tri-modality system. For this, prior to the experiment, two capillary tubes were separately filled with indocyanine green  and fluorodeoxyglucose , were inserted into the holes of the phantom. The reconstructed CT, PET, and fluorescence images were used for registration.For system testing and calibration purposes, a phantom was constructed to evaluate all three imaging modalities. The solid, cylindrical phantom with an outer diameter of 30 mm and a length of 70 mm  contained two 2 mm diameter holes of 35 mm depth. The center-to-center distance between the two holes was 10 mm. The phantom had an absorption coefficient of µa = 0.02 cmPET images were first acquired over a time period of approximately 5 min using energy and coincidence windows of 350 – 650 keV and 12 ns, respectively. Subsequently, the phantom was moved to the field of view of the CT module. A CT acquisition was performed by employing the following parameters: X-ray voltage: 80 kVp, anode current: 450 μA, and exposure time of each of the 360 rotational steps was 50 ms. Finally, the fluorescence images were collected from 36 projections evenly along with the 360° rotation, and the integration time of the sCMOS camera was 4 s at each projection angle.PET data were reconstructed after attenuation correction by using ordered-subsets expectation maximization (OSEM). A CT data were reconstructed by using a filtered back-projection algorithm. FMT data were reconstructed by the method of L1 norm regularization with modified FOCUSS (L1-FOCUSS). The obtained PET and FMT images were registered with the CT images, respectively.18F-ML-10 was performed according to a previously described approach (ML-10 (2-(5-fluoro-pentyl)-2-methyl-malonic acid, MW=206 Da) is the prototypical member of a novel family of small-molecule detectors of apoptosis. The fabrication process of approach , 23.18F-ML-10 was synthesized from its respective precursor, ML-10-mesylate, by a nucleophilic substitution reaction. For radiolabeling, 18F-fluoride delivered from a cyclotron was trapped on a quaternary ammonium cartridge, and excess 18H2O was removed. 18F was eluted with Kryptofix222 (Sigma-Aldrich) and azeotropic drying at 90°C under argon with acetonitrile resulting in dry 18F-fluoride. The precursor ML-10 mesylate in anhydrous acetonitrile was added for a 15-min reaction at 90°C. After dilution with acetonitrile, a silica Sep-Pak (Waters) was used to separate t-butyl–protected 18F-ML-10. Hydrolysis of the protecting groups was accomplished by treatment with trifluoroacetic acid (TFA):H2O (9:1) at room temperature for 15 min. Dilution with deionized water followed by semipreparative reversed-phase high-performance liquid chromatography (HPLC)  provided purified 18F-ML-10. The product eluted at a retention time of 15 min. Organic solvents were removed after dilution with deionized water, and 18F-ML-10 was trapped on a Sep-Pak C18 column (Waters) under elution with absolute ethanol. The HPLC product fraction containing 18F-ML-10 was mixed with deionized water containing 1% acetic acid (100 mL), and the mixture was passed over a Sep-Pak C18 column. Flushing with absolute ethanol (1 mL) yielded the final product. The radiochemical yield of 18F-ML-10 was 40%255% at the end of synthesis (EOS), with a collected product yield of 30%240% after purification steps (EOS). The radiochemical purity was greater than 99%, and the specific activity was greater than 40.7 GBq/mmol (EOS). The overall time required for radiolabeling was about 75 min.18F-ML-10 was injected through the tail vein to investigate the tumor apoptotic area. The specific activity of 18F-ML-10 is 51.6 MBq/g.11.1 MBq (215 µL with 0.3 mCi) of ad libitum. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Peking University institutional animal care and conducted according to the AAALAC and the IACUC guidelines. Body weight was monitored and recorded weekly.Thirty specific pathogens free (SPF) grade BALB/c-nu/nu nude mice  were housed in an environment with a temperature of 22 ± 1 °C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 hr, and were given water and food Thirty mice were used for imaging and Western Blot (WB). Among them, half of them (15) was assigned to the control group and 15 to the experimental group. The procedure of animal imaging consisted of the following steps: probe injection, probe uptake time, anesthesia procedure, and multi-modality image collection, as shown in in vivo. Briefly, two imaging probes were used for this purpose: firstly, in order to be able to investigate the necrotic tumor area, an ICG solution (8.0 mg/kg) was injected into the tail vein 24 h prior to the experiment. 8 mg/kg is a dose that takes into account both animal safety and fluorescence signal-to-noise ratio. Secondly, 11.1 MBq (0.3 mCi) of 18F-ML-10 was injected into the tail vein to detect the tumor apoptotic area. The timeline for the multi-modality imaging experiment was as follows (Semrock). The raw data were acquired under the control of a baseline clamp and processed with the help of Andor SOLIS software. Immediately after fluorescence image recording, whole-body PET/CT images were acquired.In this study, the tri-modality imaging system was used to investigate the therapeutic effect (apoptotic and necrotic tumor induction) of a new recombinant bacterial strain on mouse head and neck xenografts  follows Figure 2as described previously (6 WSU-HN6 cells were then subcutaneously injected into the mouse back (in 30 mice). When the tumor tissues reached 2-3 mm in diameter, mice were randomly assigned to a treatment or control group (10 mice in each group). The treatment group received 200 µL solution containing 1x108 B.breve-IL-24 bacteria for the treatment group, while a 200 µL saline solution was given to the control group; injections were given by the tail vein twice per week for a total of 2 weeks. Tumor size was measured twice a week post-injection by using a standard caliper tumor volume calculation method: Vtumor= (l x w²)/2. After treatments, all mice were imaged according to the in vivo imaging protocol mentioned above . Then, the mice were euthanized, and tumor tissues were dissected and analyzed by Western blot and a histopathology assay. Detailed materials and methods for Western blotting and the histopathology assay are described in the “Electronic Supplementary Material” section. Cell necrosis and apopsos could be observed by HE staining. To be specific, volume expansion and deformation of groups of cells or some tissues can be observed. Cell membrane was observed damaged. Chromatin does not aggregate and appears flocculent. Organelles are enlarged or broken. The nuclei of apoptotic cells were pyknotic and fragmented, showing blue-black color, and their cytoplasm was light red. Normal cell nuclei are uniformly blue. While necrotic nuclei appear very pale blue or disappear.The recombinant Escherichia coli–Bifidobacterium shuttle vectors pLW5 and pLW9, which include IL24 (835 bp) and green fluorescent protein  fragments, respectively, were used to obtain B. breve-IL24 (pLW5-breve) and B. breve-GFP (pLW9- breve) transformant. B.breve-IL-24 transformants were obtained through electrotransformation. Transformed strains expressing IL-24 were analyzed by RT-PCR before starting the experiment, eviously . BrieflyTumor size and real-time PCR results were expressed as mean ± standard deviation (SD). All calculations and analyses were performed using SPSS 19.0 software . A P value <0.05 was considered as being statistically significantly different. All continuous variables were first tested for normality, and then the mean difference was tested according to the group t-test. The rank-sum test was performed when continuous variables did not pass the normality test. Categorical variables were tested by using the chi-square test. A p value <0.05 was considered to be statistically significant for all statistical methods.18F-FDG within the right capillary tube is shown by a hot spot representation. Taken together, our results indicate that our multi-modality imaging system is able to provide fused images with a misalignment of less than 1 mm).Reconstructed PET, FMT, CT, and tri-modality fusion images of the cylindrical phantom are shown in in vivo. After bacteria treatment, Balb/c nude mice bearing a subcutaneous WSU-HN6 xenograft were subjected to multi-modality imaging in order to examine the apoptotic and necrotic induction within the tumor, as well as to evaluate tumor growth compared to the control group 18F-ML-10 tracer. The merged real-time images showing the apoptotic and necrotic tumor areas, including the whole-body animal structure, are shown in To obtain better subcutaneous fluorescence data, the FRI mode was used in the animal study. Each rotational angle of the acquisition was recorded so that the FRI image could be successfully projected and merged with the CT images. The results indicated cell death in the center part of the xenograft WSU-HN6  was injected. Results showed higher tumors grew in the control group compared to B.breve-IL-24-treated mice, which suggests that the B.breve-IL-24 strain can inhibit tumor growth over time  in B. breve-IL24 was smaller compared with the B. breve-GFP group.To further evaluate the antitumor effect of B.breve-IL-24 Interleukin-24 (IL-24) is a novel tumor suppressor cytokine that selectively induces apoptosis in a wide variety of tumor types. To further substantiate an activation of the apoptosis pathway, we analyzed the effect of B.breve-IL-24 on caspase-3 activation in tumor tissues. We found that B.breve-IL-24 induced activation of caspase-3 and Bim in tumor tissues, the results could be found in our previous study .in vivo (The PET/CT scanning results of the tumors treated with B.breve-IL-24 showed a significantly higher apoptosis induction as detected by the higher radioactivity (135.35 µCi) compared to the control group (20.09 µCi) in vivo .Finally, the necrotic tumor area was analyzed by bright field microscopy after histopathologic tissue staining. In contrast to the apoptosis induction results, a larger necrotic tumor area was observed in the control group  with an anatomic imaging modality (CT). In this study, we described the system’s technological development, the optimization strategy of the imaging protocols, and the system performance in vivo. On the one hand, PET provides robust in-depth detection capabilities, on the other hand, it requires the administration of a radioactive contrast agent. In any case, applications being able to detect tissues apoptosis would benefit from any imaging modality, no matter of which type. Therefore, current strategies in tissues apoptosis imaging rely on multi-modality approaches that combine the strengths and capabilities of different imaging techniques , respectively, have been developed –28. Howechniques . Howeverchniques .18F-ML-10, was used to further investigate apoptosis induction in malignant cells. In addition, the CT scan was used to examine the anatomical information of the animal body and bone structures. To conclude, our data suggest that the combination of these imaging tools might provide more accurate and comprehensive information about tumor behavior and the therapy success in vivo.The three modalities (PET/CT/FMI) were integrated in the same animal bed and images were taken in a parallel scan leading to a more accurate registration compared to software-based methods used in single (separate) modality scans. Consequently, this integrated system offers a lower mouse throughput-to-cost ratio, a shorter scan duration and less motion artifacts. Therefore, the system provides synergistic advantages compared to single imaging modalities. In this study, a near infra-red fluorescent dye, ICG, was used in FRI, in order to provide information about tumor cell death (necrosis) while a novel PET tracer, in vivo; usually only 3-5% of tumor cells are considered in the growth fraction while the rest 95% of tumor tissue is hypoxic to some degree  for small animals was developed. This system integrates structural, functional, and molecular imaging with a therapeutic approach. Furthermore, this new translational platform combines common optical techniques and nuclear medicine methods and represents the ultimate multi-modality platform in biomedical research, thus offering a powerful diagnostic approach to investigate the tumor behavior and therapy in real-time. Moreover, we have established and validated the use of a new recombinant bacteria B.breve-IL-24 gene for cancer therapy The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Peking University ethic committee.YK, XZ and SL designed the idea and finished the manuscript. ZX contributed to designing the three-modal image system. IV, LW and KZ performed the biochemistry and immunology experiments. ZP helped to elaborate the content about cell apopsis and checked the expression. XZ and SL analyzed the data. YK, ZX and QR supervised. YK, XZ, SL, IV and LW revised the manuscript based on reviewers' comments. All the authors had read the manuscript and helped to improve the expression. All authors contributed to the article and approved the submitted version.This work was supported by The National Key Instrumentation Development Project (2011YQ030114), the National Basic Research Program of China , and the National Natural Science Foundation of China (11104058).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: This study aimed to estimate the impact of the COVID-19 lockdown on fitness performance among Chinese college men during the pandemic period and to explore how fitness changed with a different college grade. Methods: We conducted repeated measures of 1000 m running and pull-up testing on students from one university in China before and after the lockdown. A total of 7107 (age 19.21 ± 1.17 yr.) male students who completed the same 1000 m running and pull-up testing in 2019 and 2020 were included in the analysis. Results: The paired t-test result indicates a reduction in 1000 m running and pull-up performance by 10.91%  and 23.89% , respectively. Interestingly, college men in the 2017 grade (the third-year college men) had more decreases than in the 2019 grade (the first-year college men). The 1000 m running performance was decreased by 14.43% and 6.48% in the third- and the first-year college men, respectively. The pull-up performance was decreased by 39.11 % in the third-year college men while increased by 10.98% in the first-year college men. Conclusions: The COVID-19 lockdown reduced 1000 m running and pull-up performances among Chinese college men. The reduction varies by grade and it seems to be particularly seriously decreased for the third-year college men while being modest for the first-year college men. Public policy was urgently needed to improve Chinese college men’s fitness performance after the lockdown. On 11 March 2020, the respiratory disease caused by COVID-19 was declared a pandemic by the World Health Organization (WHO) and a national emergency in China. To date (29 June 2022), 4,249,438 confirmed cases of COVID-19 with 21,073 deaths were reported to the National Health Commission of the People’s Republic of China [To slow the spread of COVID-19, school closure and home confinement measures were necessary. However, they also created various effects in different domains, such as physical inactivity, weight gain, irritability, depression, and anxiety ,5,6,7,8.To prevent the adverse effects of COVID-19 on physical fitness among adolescents, many schools and colleges forced students to participate in web-based physical education (PE) classes at home instead of on the playground . AlthougFurthermore, reducing PA levels and physical fitness during the COVID-19 pandemic would probably cause health problems for college students. Globally, nearly 80% of adolescents are insufficiently active ,19. It iDespite the previous work, two main gaps exist in the earlier studies. First, although previous studies have stated the decline of PA levels during the COVID-19 pandemic, which might lead to the potential negative impact on fitness ,30, few To address this knowledge gap, this study aims to estimate the effects of the COVID-19 pandemic on Chinese college men’s fitness performance (1000 m running and pull-ups). Furthermore, this study also intends to investigate potential differences between college men of different grades. In this case, the novelty of this study was performing repeated tests on a large sample of 7107 people in 1000 m running and pull-up performances pre- and post-COVID-19. This study would provide data to contrast the effects of the COVID-19 pandemic on physical fitness in a large sample among college men in China and make a classification of the subjects by grade. It would provide novel insight into the effects of COVID-19 on the physiological states of the population.A total of 7107 college men completed the physical fitness testing in 2019 (pre-COVID-19 lockdown) and 2020 (post-COVID-19 lockdown). The college men were from three different grades: 2171 college men were from the 2017 grade (college men who entered college in 2017); 2437 college men were from the 2018 grade (college men who entered college in 2018); 2500 college men were from the 2019 grade (college men who entered college in 2019). Participants who completed the 1000 m running and pull-up performance tests in 2019 and 2020 were included in the analysis of this study.At Tsinghua University, China, all college students must participate in physical fitness testing every autumn semester with exceptions for special conditions, such as illness or disability. According to the National Student Physical Health Standard (NSPHS) in China , the phyTrained teams took the 1000 m running and pull-up testing pre- and post-COVID-19 lockdown. The period of the COVID-19 lockdown for Tsinghua University students was from mid-Jan 2020 to late-August 2020. During the lockdown period, all students stayed at home and no longer had access to college-based physical activities. After late August, the college students returned to school and engaged in college-based physical activities. In this context, the pre-COVID-19 testing was performed from 9 September to 10 November 2019, and the post-COVID-19 testing was performed from 19 October to 14 November 2020. The testing was carried out from 8:00 am to 12:15 am and 1:30 pm to 6:40 pm. The testing procedures and measurements were the same in the pre- and post-COVID-19 testing. None of the participants tested positive for COVID-19 before the post-testing. Tsinghua University ethics review boards approved the study (IRB #201253400). Participants provided written informed consent before the testing.The 1000 m running testing was conducted on the playground of Tsinghua University and measured by the time of a 1000 m race recorded in seconds. Before the 1000 m running testing, participants were asked to warm up under the guidance of our trained teams. During the testing, participants were asked to wear a vest containing a timing chip. The tester could automatically measure participants’ running time using an infrared sensor. Participants were asked to finish the 1000 m running as fast as possible, and their running time in the range of 00:00–16:66 (min:sec) was recorded and included in this study. Each participant only performed the 1000 m running testing once. The tester used the running tester of Tongfang Health Fitness Testing Products 5000 series  . The tesThe pull-up testing was conducted in the fitness square of Tsinghua University. Before the pull-up testing, participants were asked to warm up under the guidance of our trained teams. After warming up, we measured the pull-up testing by the repetitions of the completed required action. Participants need to pull themselves up until their chin clears the bar with an overhand grasp and finish by lowering the body until they fully extended their arms and shoulders. The number of repetitions they performed the pull-up action until they were fully exhausted were be recorded. Each participant performed the pull-up testing one time. Their actions in the range of 0–100 repetitions were recorded and included in this study. The tester used was the pull-up tester of Tongfang Health Fitness Testing Products 5000 series  . The test-test was conducted to compare mean differences in the same sample before and after the COVID-19 lockdown. In the analysis, participants’ 1000 m running and pull-up performances before and after the lockdown were performed by paired t-test analysis. We performed paired t-test using bootstrapping and stratified age and BMI variables as covariates. In addition, we analyzed the effect size of Cohen’s d  and compared the differences among the measurement points in the 2017 grade college men, the 2018 grade college men, and the 2019 grade college men. The fitness changes compared with the 2019 baseline as the reference. For example,To sum up and compare the characteristics of the overall sample, descriptive statistics of this research used means and standard deviations and percentages. One-way ANOVA was used for the comparison of the baseline characteristics of 2017, 2018, and 2019 grade college men. We analyzed participants’ physical fitness level changes before and after the lockdown comparison. We performed a two-way ANOVA to examine the groups and time as factors to assess the interaction between groups and time. A paired p < 0.05.We also analyzed a subgroup of the fitness change differences among the 2017 grade men, 2018 grade men, and 2019 grade men. We performed all the statistical tests using IBM SPSS Statistics 28.0 . The level of statistical significance was set at 2) and the mean BMI of participants from 2017, 2018, and 2019 grade was 22.90 ± 3.62 (kg/m2), 22.31 ± 3.58 (kg/m2) and 21.94 ± 3.46 (kg/m2), respectively. The mean level of 1000 m running performance of all the participants was 4:05 ± 0:28 (min:sec) and the mean level of 1000 m running performance of participants from 2017, 2018, and 2019 grades was 4:13 ± 0:29 (min:sec), 4:03 ± 0:26 (min:sec), and 4:00 ± 0:27 (min:sec), respectively. The mean level of pull-up performance of all the participants was 7.42 ± 7.21 (repetitions) and the mean level of pull-up performance of participants from 2017, 2018, and 2019 grades was 9.04 ± 7.30 (repetitions), 9.06 ± 7.59 (repetitions), and 4.35 ± 5.57 (repetitions), respectively.p < 0.001) in the 1000 m running performance  and the pull-up performance , pre- and post-COVID-19 lockdown.p < 0.001).p < 0.001) and the effect size was large (d = 0.92). Furthermore, the 1000 m running level decreased for 2017, 2018, and 2019 grade men by 14.43% (p < 0.001), 12.09% (p < 0.001), and 6.48% (p < 0.001), respectively. The effect size for the 2017 and 2018 grades was large  and the effect size for the 2019 grade was medium (d = 0.73)  see .p < 0.001) and the effect size was small (d = 0.33). In addition, the pull-up levels decreased for the 2017 and 2018 grade men by 39.11% and 28.31%, respectively. The effect size for the 2017 and 2018 grades was medium (d = 0.78) and small (d = 0.39). Interestingly, the results show that the 2019 grade men’s pull-up levels increased by 10.98% and the effect size was small (d = 0.14)  see .This study assessed the impact of the COVID-19 lockdown on Chinese college men’s 1000 m running and pull-up performances by grade. We found that Chinese college men significantly decreased their 1000 m running and pull-up performances due to the COVID-19 lockdown. Furthermore, we found that participants’ fitness was differently decreased by grade. Compared with the 2019 grade men, it seemed to be a more serious decrease for the 2017 and 2018 grade men. Interestingly, we also found that the 2019 grade men’s pull-up testing results performed better after the school closure. To the best of our knowledge, this is the first finding that examines the impacts of the COVID-19 lockdown on 1000 m running and pull-up performances for Chinese college men by grade.p < 0.001) and 23.89% (p < 0.001), respectively. This finding was consistent with a previous study showing that adolescents decreased their fitness levels after the COVID-19 lockdown [In this study, we provided evidence to confirm the negative impact of the COVID-19 lockdown on the 1000 m running and pull-up performances among college men living in China. In our study, we found that college men’s 1000 m running and pull-up levels totally significantly decreased by 10.91% (lockdown ,40,41. Plockdown . SimilarMoreover, this finding was inconsistent with a recent study revealing that college men’s pull-up performances increased during the lockdown . This stInterestingly, our study found that the impact of COVID-19 on physical fitness among college men varies by grade. Although the 2017 and 2018 grade men significantly declined on the 1000 m running and pull-up performances, the 2019 grade men increased their pull-up performance by 10.98%. A possible explanation for this finding was PE courses could play a crucial role in maintaining and improving college students’ pull-up performances. In our study, before the COVID-19 lockdown, the college men’s pull-up mean levels of 2017, 2018, and 2019 grades was 9.12, 9.29, and 4.35 repetitions, respectively. Thus, we could infer that before the COVID-19 lockdown, college men in 2017 and 2018 grades had experienced more than one year’s campus PE training, while college men in 2019 grades just experienced a short period of campus PE training. Our participants might have a stronger fitness in college before the lockdown and are more likely to be affected by the school closure.Several factors could contribute to the decline of college men’s physical fitness. First, a possible reason for the decrease in college men’s physical fitness was that indoor or outdoor PE courses had been replaced by web-based PE courses . Due to The strength of our study is that this is a repeating test on the same sample and a large sample in 1000 m running and pull-up levels of Chinese college men by grade before and after the COVID-19 lockdown. Nevertheless, our study also has limitations. Firstly, although college men’s cardiorespiratory fitness and muscular strength are usually assessed by 1000 m running and pull-up performances in China, the 1.5 mile and 12 min walk/run tests, which represent useful alternatives for estimating cardiorespiratory fitness , should COVID-19 decreased Chinese college men’s 1000 m running and pull-up performances. The impact varied by grade and it seemed particularly serious for the third-year college men while being modest for the first-year college men. Public policy was urgently needed to improve Chinese college men’s fitness performance after the lockdown.